Advances in engineering have made possible the production of commercial quantities of therapeutically useful proteins which heretofore have been too complex or expensive to manufacture through traditional biochemical processes. Manufacturing is accomplished by cells that are engineered to produce a desired protein and can be grown in bioreactors under controlled conditions. The technology used involves either the fermentation of microorganisms which have been altered through recombinant DNA techniques or the culturing of mammalian cells which have been altered through hybridoma techniques. The cells are suspended in a broth which contains the salts, sugars, proteins, and various factors necessary to support the growth of particular cells. The desired protein may be either secreted by the cells into the broth or retained within the cell body.
Other proteins of interest can be prepared using alternative means, such as by genetically altering an animal species in order to provide the animal with the capability of producing the desired protein. For example, certain proteins can be produced by transgenic cows and harvested from their milk.
The separation, or purification, of these proteins from a heterogenous mixture has proven to be a formidable task for at least the following reasons: the desired protein often represents a small percentage of total protein; the broth or other liquid to be processed may contain significant quantities of cell debris and other particulate contaminants; pyrogens, pathogens, toxins, and other contaminants may be present in high concentration and must be removed; and the desired protein must be separated from the heterogenous protein solution without denaturing it.
As a result of these factors, extensive downstream processing has been necessarily used to yield high quantities of purified protein. Such downstream processing includes the many stages of processing that take place subsequent to the production of the protein of interest including, for example, centrifugation, cell disruption, mechanical sieving, microfiltration, ion-exchange, cross-flow filtration, affinity separation, sterilization, purification, and packaging. The downstream processing represents a major cost in the production of bioprocessed proteins. Thus, a method which efficiently provides high yielding protein separation by reducing the number of processing steps, and which can be used on an industrial scale, would further the successful commercialization of biotechnology.
It is known that many compounds complex with ligands such that those compounds may be isolated by a technique known as affinity separation. See generally "Chromatography, Affinity," Kirk-Othmer Encyclopedia of Chemical Technology, 6, 35-54 (John Wiley & Sons: New York, 1979). This method of separation involves three phases: (i) an adsorption phase, wherein the desired compound, such as a protein, from a heterogenous mixture forms a complex with a chemical functionality, such as a ligand, bound to an insoluble substrate, such as a polymeric or glass bead, (ii) a washing phase wherein the bulk of the solution is washed away along with contaminants loosely bound to the insoluble substrate, and (iii) an elution stage wherein an eluant breaks the complex between the desired compound and the ligand bound to the insoluble substrate so as to release the desired compound. The insoluble substrate with the bound ligand, which is referred to as the affinity particles, may be washed and reused, and the method may be repeated numerous times.
The affinity particles are typically selected based upon surface area per unit mass. For example, while nonporous spherical beads of 100 microns diameter may provide a surface area of 0.06 m.sup.2 /g of beads, similar beads of 1 micron diameter would provide a surface area of 6.0 m.sup.2 /g of beads, and beads of 0.1 micron diameter would provide a surface area of 60 m.sup.2 /g of beads. Clearly, smaller affinity particles provide a larger surface area per unit weight and are more desirable. In affinity separation, it is generally desirable to utilize affinity particles which have a surface area of greater than about 20 m.sup.2 /g. Thus, if one desires to use nonporous affinity particles in an affinity separation process, such affinity particles must have a diameter in the submicron range.
The most widely practiced form of affinity separation is affinity chromatography. Affinity chromatography utilizes a column of tightly packed affinity particles through which the fluid (e.g., liquid feed stream) containing the target compound, such as a protein, is forced under pressure. The hydrodynamics of a packed or fixed bed system such as this is governed by the Ergun equation. Chem. Eng. Prog., 48 (1952). The Ergun equation, which is used when the bed experiences laminar flow of fluid, demonstrates that the pressure drop across the bed is inversely proportional to the square of the particle diameter. When calculated, the pressure drops of systems which use submicron affinity particles are in the order of one million times greater than those packed with 100 micron particles. This effect has heretofore prevented the use of such small affinity particles in affinity separations. Consequently, the typical bed utilizes affinity particles of about 100 microns in size to maintain an acceptable pressure drop.
Attempts have been made to overcome the problems associated with nonporous affinity particles with respect to providing a high surface area per unit weight. In particular, the nonporous affinity particles have been substituted with porous affinity particles. In typical applications, porous beads of about 100 microns in diameter provide a surface area in the range of about 40 m.sup.2 /g per bead under moderate pressure drop conditions.
The use of porous affinity particles, however, creates additional problems which are not experienced by nonporous affinity particles. For reasons not entirely understood, but perhaps relating to plugging and diffusional resistance, only the outer about 5 percent of the porous region of the affinity particle is generally accessible to the substance sought to be separated. This results in the effective surface area of the porous affinity particle being much less than the total theoretically available surface area. While the pretreatment of the feed stream could prevent at least a portion of the plugging and diffusional resistance of the porous affinity particles, such pretreatment of the feed stream involves a costly and time consuming series of steps. Further, each additional processing step serves to increase the loss of the target compound and thereby reduce productivity.
Moreover, because the majority of the total surface area in the porous affinity particles is interior of the surface of the affinity particles, mass transfer effectively controls the separation process, thereby resulting in time consuming and laborious separations. Further, the pores of the affinity particles can act as traps for unwanted compounds and debris and can eventually cause contamination of the desired compound. Also, as the pores in the affinity particles gradually plug, the effectiveness of the affinity particles is decreased with each succeeding cycle. In addition, the mechanical strength of the affinity particles is reduced due to the presence of the pores, which results in poor abrasion resistance and mechanical stability and may result in affinity particle collapse at high pressures. The porous affinity particles also swell as they are packed in a bed, which requires the adjustments to be made for this phenomenon.
Another consideration of column techniques which is affinity particle-independent is that, in order to achieve high flow rates, the column must have a large capacity. Tall columns, however, result in such high pressures at the lower zone of the columns that they deform the affinity particles and reduce throughput. The industry has attempted to overcome this problem by designing columns which are short and rather wide. This compromise, however, is not entirely satisfactory, since efficiency is greatly reduced due to flow problems like channeling and departure from plug flow.
Columns also exhibit a concentration wave phenomenon that results in a low effective utilization of adsorbent. Specifically, as feed passes through the column, only a small zone of affinity particles is utilized in a particular time frame since the entrance zone is already saturated with the target compound. This results in a concentration wave phenomenon where only the wave front interacts with the adsorbent at a given moment.
In order to overcome the disadvantages inherent in fixed beds and the problems which arise when porous affinity particles are used therein, researchers have turned their attention toward moving bed affinity separation systems.
A fluidized bed is one type of moving bed affinity system. In such a bed, the feed stream is pumped up from the bottom of a column, causing the affinity particles to act like a fluid. As a result, fluidized beds do not exhibit concentration wave characteristics. Moreover, all the affinity particles are exposed to the feed stream simultaneously. Fluidized beds offer higher productivity than columns as a result of the efficient utilization of the affinity particles and increased throughput. Further, these beds can accept a higher level of particulate debris and still function relatively efficiently as compared to fixed bed systems.
A drawback with fluidized beds, however, is that efficiencies limit both the size and density of the affinity particles that can be used in such systems, i.e., it is necessary to maximize density and optimize size. This is because, generally, a major part of the separation process is controlled by the settling velocity of the affinity particles. Thus, the higher the density of the affinity particles used, the faster the affinity particles will settle, thereby enabling the use of higher fluidization velocities. An increase in the fluidization velocity will result in a higher throughput and, therefore, higher efficiency, until the upper limit of the rate of kinetics is reached. While the rate of kinetics can be increased by reducing the size of the affinity particles (thereby increasing the total available surface area), the increase of the rate of kinetics also has the negative effect of limiting the fluidization velocity that can be achieved, thereby lowering efficiency. These competing considerations require that compromises be made with regard to the size and density of the affinity particles, and these compromises prevent this type of system from reaching higher efficiencies.
Another type of moving bed system, referred to as a stirred tank system, also offers significant benefits over fixed bed columns. In a stirred tank system, affinity particles are kept suspended in a tank by mechanical means. This mixing action allows the suspension to behave like a homogeneous fluid, thereby exposing all the particles to the target compound simultaneously. This process can therefore be designed as a loop, with a significant increase in productivity. The affinity particles in such a system are preferably sufficiently buoyant to allow for a stable suspension in the process fluid. Since there is less packing in this system, the preferred diameter of the affinity particles is typically smaller than in a column or fluidized bed, which allows for an increase in surface area. A major limitation of stirred tanks is that the filters used for the wash and elution stages foul as a result of the crude nature of the feed stock. Thus, the feed stream must typically be pretreated, thereby adding an additional processing step and reducing the overall system efficiency.
In each of these various conventional affinity separation processes, there are multiple processing steps. While each step may have an efficiency or product yield of 80-90%, the existence of only a few processing steps can easily reduce the overall system efficiency and product yield to 50% or less. With each additional processing step, the overall system efficiency is even further lowered.
Thus, there exists a need for a method which avoids these many problems inherent in the existing affinity separation processes and provides a more efficient means for isolating and separating compounds, such as proteins and the like, from a fluid, e.g, a dilute liquid feed stream. The present invention provides such a method. The present inventive method involves an affinity separation process with fewer processing steps which can be carried out in a relatively short period of time and with an increased overall efficiency to remove a desired compound from a fluid. Moreover, the present inventive method overcomes the problems of fouling as regarding both the affinity particles and filtration medium which hamper the kinetics and flow-through of the system, without the need to pretreat the fluid containing the desired compound. Further, a wide variety of affinity particles varying in both density and size, including small affinity particles with a high surface area per unit of weight can be used successfully in the affinity separation method of the present invention, thereby enabling the system to be customized to achieve greater efficiencies for particular end uses. These and other objects and advantages of the present invention, as well as additional inventive features, will be apparent from the description of the invention provided herein.